Project Summary/Abstract Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa, a diverse group of parasites that cause various diseases in humans, such as malaria and cryptosporidiosis. T. gondii is ubiquitous worldwide, estimated to infect one-third of the human population, and is a public health concern in immunocompromised and pregnant individuals. The acute phase of infection is characterized by the tachyzoite, a fast-replicating life stage that disseminates throughout the body of a warm-blooded host. The chronic phase of infection is characterized by the bradyzoite, a slowly- replicating life stage which encysts in muscle and neural tissue via the formation of a cyst wall within a host cell. Bradyzoite biology is poorly understood; the exact composition of the cyst wall, how bradyzoites persist within host cells indefinitely, or how bradyzoites manipulate host cell function is unknown. Previous studies have demonstrated the secretion of several protein effectors by tachyzoites into their host cells across the parasitophorous vacuole, the site where parasites replicate within host cells. These include the proteins GRA16, GRA24, GRA28, and TgIST, most of which have been shown to localize to the host cell nucleus and directly interact with host cell proteins, altering host cell signaling cascades and transcription. Whether these proteins, or other unidentified secreted proteins, cross the cyst wall and enter bradyzoite infected host cells has not been previously explored. In this proposal, the first aim will be to determine the localization of GRA16, GRA24, GRA28, and TgIST after bradyzoite differentiation in vitro by epitope tagging secreted effectors at their endogenous loci and in vivo in the mouse brain using an optical clearing approach. In the second aim, the role of TgIST in bradyzoite infected cells will be investigated by knocking out and complementing the TgIST gene in the parasite and subsequently determining the inhibition of STAT1 mediated transcription in bradyzoite infected host cells. In the third aim, proximity-based biotin labeling enzymes will be fused to MYR1, a protein implicated in parasite protein export into the host cell. This will allow the identification of secreted effectors during the bradyzoite stage by mass spectrometry of biotinylated proteins following validation of positive hits. The discovery of secreted effectors by bradyzoites into host cells would challenge a paradigm in the field of Toxoplasma research, where the bradyzoite has classically been viewed as an inert but transmissible stage of the parasite. This will lead to further studies on understanding how bradyzoites alter host cell function and maintain persistency in their hosts.